Collagen fibril and neutral salt composition



United States Patent Ofifice 3,374,103 Patented Mar. 19, 1968 ABSTRACT OF THE DISCLOSURE An improvement in the dry knot tensile strength and dry straight tensile strength of an extruded collagen suture is obtained when from about 0.01 percent to about 0.07 percent of a water soluble salt is added to the dispersion of acid-swollen collagen fibrils prior to extrusion.

This invention relates to a dispersion of swollen collagen fibrils in an acid solution containing an added electrolyte and to a process for obtaining such a dispersion. The collagen dispersions of the present invention are useful in the manufacture of absorbable sutures and surgical aids in the form of films, filaments, strands, tubing and sponges.

The extrusion of a homogeneous dispersion of swollen collagen fibrils to form collagen strands that may be used as surgical sutures is described in US. Patent No. 3,114,372 and No. 3,114,593. An important aspect of the process therein described is the preparation of a homogeneous dispersion of swollen collagen fibrils that is suitable for extrusion and subsequent processing. In particular the integrity of collagen fibril structure as it exists in its native state must be maintained throughout the steps of comminution, enzyme treatment, acid swelling, filtration, extrusion, stretching, tanning and drying.

Suitable dispersions of swollen collagen fibrils prepared in accordance with the aforesaid invention may have a solids content of the order of 0.72% to 1.1%. However,- the utilization of such collagen dispersions having the low solids content indicated above and in which the collagen material has been reduced to particles of fibrillar size introduces difficult problems in treating and handling the extruded filament from the moment it leaves the orifice of the spinnerette until it has been coalesced with other filaments and dried to the ultimate size at which it is to be later used. A collagen dispersion having a sodids content of about 1% is highly viscous and dilficult to extrude. Moreover, the viscosity of such dispersions interferes with the parallel orientation of the collagen fibrils upon extrusion.

It is an object of the present invention to provide a low viscosity dispersion of swollen collagen fibrils that may be extruded to form a collagen strand of improved tensile strength.

In accordance with the present invention, it has now been discovered that the addition of very small amounts of an electrolyte to a homogeneous dispersion of collagen fibrils causes a marked reduction in the viscosity of the dispersion and thereby facilitates filtration, pumping and extrusion. Collagen strands prepared from dispersions that contain a small amount of an electrolyte have better tensile properties than those obtained from dispersions containing no added electrolyte. v

The electrolyte employed may be any water-soluble salt of a monovalent, divalent or trivalent metal, such as sodium nitrite, sodium chloride, potassium acetate, potassium sulfate, copper sulfate, chromic chloride; an ammonium salt, such as ammonium nitrate, ammonium sulfate, ammonium chloride, etc, or a quaternary ammonium salt, i.e. benzethonium chloride, having the structure:

l nens o1-.Hio

While the present invention is not to be limited to any particular theory of operation, one may speculate that the addition of an electrolyte to an acid dispersion of swollen collagen fibrils causes a reduction in the swelling of the fibrils as evidenced by a decrease in the viscosity of the system. Specific interactions between the salt ions and charged groups of the proteins and/or shielding of the charged protein groups by the salt ions are largely responsible for this effect. It may be hypothecated that the reduced swelling of the collagen fibrils in the.presence of added electrolyte retards the degradation and morphological changes that can take place in collagen fibrils that are suspended in acid solution. This effect is more pronounced at lower pH and elevated temperature but occurs to some slight degree upon long storage at room temperature.

The raw material for the dispersion of this invention is mammalian tendon. Whales are a large source of collagen, and whale tendon is a satisfactory starting material. Pork, sheep and beef tendons are also satisfactory. The best results to date have been obtained using the deep flexor tendon of cattle,

The cattle dispersions of the present invention may be prepared by the general procedure described in US. Patent No. 2,920,000. Beef tendon as shipped from the packing house and received, has been frozen to prevent deterioration and must be thawed to permit cleaning the tendon of fat, superficial non-collagenous protein, and other extraneous matter. The clean tendon is then sliced to a thickness of about 10 to 25 mils. Thicker slices swell slowly in aqueous acid solutions and are diificult to disperse. Thinner slices disperse readily but the dispersion, when extruded, has poor tensile strength. Preferably, the tendon sections are sliced across the major axis as lengthwise slicing seems to result in a slower swelling.

The sliced tendon is next treated with an enzyme solution to solubilize the elastin network which encircles the, native collagen fibers and ties them together. By this treatment, the greater part of the elastin is solubilized and can be removed. Proteolytic enzymes from eithen plant or animal sources may be employed to advantage. Pancreatin is an enzyme that is elfective in removing elastin. Enzymes derived from plants, such as ficin, are also useful. Another enzyme that will perform this function is one prepared by extracting commercial malt diastase (U.S.P. IX) with water. The tendon-enzyme mixture is stored at room temperature for to hours.

After the enzyme treatment, the tendon slices are inactivated with hydrogen peroxide and washed with water. Soluble proteins and lipids may be removed by treating the slices with a dilute aqueous solution of a chelating agent, such as ethylenediamine tetrasodium tetraacetate. Following this treatment, the tendon slices are washed again to remove residual traces of the chelating agent.

The cleaned tendon slices at this point contain a high percentage of purified collagen associated with material that does not swell in an acid solution. The next step is to swell this collagen in an acid solution to form a homogeneous dispersion of collagen fibrils, but it is most important that during this step the individual slices of collagen not be permitted to coalesce. As collagen swells, it becomes sticky and, if the individual collagen slices are permitted to stick together, the interior of the conglomeration will not have contact with the swelling solution. Therefore, to obtain a homogeneous fiber dispersion in a practical time, it is most desirable to prevent coalescence of the individual tendon slices. A dispersion kettle having a paddle positioned off center may be used to minimize lump entaglement. In the dispersion kettle, the collagen slices are slowly stirred in the aqueous acid solution. The collagen slices absorb the acid solution with swelling.

Temperature becomes a critical factor after addition of acid to the tendon slices as the collagen is degraded in the presence of acids above about C. For this reason, all processing subsequent to the acid addition should be carried out at temperatures below about 25 C.

The swelling solution may be an aqueous solution of an organic acid characterized by a pKa value greater than 2.40; i.e. acetic acid (PKOL=4.76). Methanol may be added to the aqueous acid solution if desired.

In general, an acid content of from about 0.20 to about 1 percent of the total weight of the solution is used. Such solutions have a pH of about 2.3. The electrolyte preferably is dissolved in this acid solution before the swelling step.

When the collagen dispersion is to be used in the extrusion of filaments, the amount of tendon present in the swelling solution is preferably about 1 percent. As previously mentioned above, a dispersion of collagen fibrils that has a solids content below about 0.7 percent is difficult to spin. On the other hand, a concentration of collagen fibrils. greater than about 1.1 percent results in a dispersion that is more difficult to extrude through a spinnerette. It is important that a collagen fibril dispersion which is to be extruded be homogeneous, as a small change in the solids concentration of the material being extruded will result in large cross-sectional variations in the final product.

After most of the swelling has taken place in the dispersion kettle, the dispersion is homogenized by repeated passes through a stainless steel rotary metering pump and stainless steel series-connected jets having orifices of about 5,0 mils and 40 mils, respectively.

Stirring is continued during homogenization. Best results are obtained with a slow agitation during the swelling stage, intermittent agitation at the beginning of homogenization, and higher speed agitation near the end of the homogenization.

The homogenizing pump employed in this process is a rotary pump (such as a Zenith pump) that has been modified by milling about 0.003 inch from the circumference of the gear teeth. The intake and exit from the pump are connected to the dispersion kettle by a stainless steel conduit which is capable of withstanding the high pressure. v g

The dispersion after homogenization still contains fibers of unswollen non-collagenous material which must be removed. This is most readily accomplished by forcing the dispersion under pressure through a leaf filter which retains the unswollen non-collagenous material.

The dispersion of collagen fibrils after filtration may be deaerated under vacuum and is then ready for storage. It stored at 5 C., or below, the dispersion will remain substantially unchanged for periods in excess of two or three Weeks.

Throughout the specification and the examples which follow, all quantities are expressed in parts by Weight.

Example I That portion of the deep fiexor tendon of cattle designated in FIGURE 2 US. Patent No. 2,920,000 as the C and D sections are cleaned of fat, superificial noncollagenous protein, and other extraneous matter and sliced on an electric meat-slicing machine (rotary knife) in the frozen condition. The tendon sections are sliced perpendicularly to their longitudinal axis to a thickness of about 11 mils. An aliquot sample of the tendon slices is analyzed; the dry solids amount to 36.97%.

The sliced tendon is next treated with an enzyme solution to solubilize elastin and remove non-collagenous protein. The enzyme solution is prepared by dissolving 0.15 part of ficin and 3.75 parts of ethylenediamine tetrasodium tetraacetate in 750 parts of water. Seventy= five parts of the sliced tendon is immersed in this solu tion which is stored at room temperature overnight. Then, 2.25 parts of 30% hydrogen peroxide is added to destroy any residual ficin.

To this mixture of tendon slices in about 750 parts of water is added an additional 2244 parts of water and 87 parts of cyanoacetic acid. The swelling solution is cooled to below 25 C. This mixture is stirred in a dispersion kettle at about 60 r.p.m. It is important that the remain= ing steps in the process be carried out at temperatures below about 25 C., and that the temperature of the collagen dispersion not be allowed to exceed this ternperature.

Stirring is continued for about 3 hours, during which time the individual collagen slices are swollen. The dis.- persion is then homogenized by repeated passes through the stainless steel rotary metering pump, and stainless steel series-connected jets having orifices of 50 mils and 40 mils, respectively. During the homogenization, the stirrer in the dispersion kettle is operated intermittently.

The pressure on the high pressure side of the homogenization jets falls to 70 pounds per square inch and remains constant after 3.5 hours, indicating substantially complete homogenization. The dispersion is then forced through a leaf filter.

The dispersion of solvated collagen fibrils so obtained analyzes 1.05% solids and has a pH of 2.52. The dispersion is extruded through a spinnerette into an acetone dehydrating bath as described in Examples X and XI of US. Patent No. 3,114,372't0 form a size 1 chrome-tanned suture.

A suture of the same size is prepared by the same procedure except that 0.03% ammonium nitrate is added to the solution of 2244 parts of water and 5.87 parts of cyanoacetic acid prior to acid-swelling of the collagen slices. A comparison of the tensile strength of this suture prepared from a collagen dispersion containing 0.03% ammonium nitrate and the suture prepared from a collagen dispersion containing no added electrolyte. It is summarized in Table 1.

TABLE 1 Diameter Tensile Strength Suture in Mils Remarks Dry Wet Knot Knot Example IA.... 22.29 9. 82 7. 94 N salt added. Example IB.... 22.27 10. 72 8.42 0.03% NH N03 added.

Example II TABLE 2 Tensile Strength Tensile Strength in Pounds in Pounds Finished 2/0 Chromle Strand Dry Wet Dry Dry Wet Straight Straight Straight Knot Knot Additive:

0% NaCl 11. 9 3.5 1115 5.8 3. 6 v 0 005% NaOl... 11.9 4.8 10.9 5. 9 3.8 0 01% NaCl.... 13. 8 5. 3 14. 5 6.9 4. 0 0.03% NaOL-.. 12. 3 3. 1 11. 7 6.3 3. 6 0.05% NaCl. 13. 9 5. 3 10.4 6.4 3. 7 Pilot Plant Preparation:

0.05% NaCl 13. 7 4.2 11. 9 6.5 4.4

1 Average of six random samples obtained from pilot plant production Example III Collagen strands, size 2/0, are prepared by the indirect spinning process as described in U.S. Patent No. 3,114,- 372. The strands are tanned with 2,4-dihydroxybenzoic acid (1% solution), aluminum formoacetate (1% solution), and formaldehyde (0.25% solution). The etfect of the addition of sodium chloride to the collagen dispersion is shown in Table 3.

TABLE 3 Dry Dry Wet Straight Knot Knot Additive:

0% 10.4 5. 2 4. 2 0.05% NoOl 12.6 5.9 3.8

Example IV Collagen strands, size 2/ 0, are prepared by the indirect spinning process as described in U.S. Patent No. 3,114,- 372. The effect of the addition of benzethonium chloride to the collagen dispersion is shown in Table 4.

Collagen sutures are prepared by the indirect spinning process as described in U.S. Patent No. 3,114,372. The effect of the addition of ammonium nitrate to the collagen dispersion is shown in Table 5.

TABLE 5 Tensile Strength in Pounds Strand Dry Dry Knot Straight Additive:

0.01 NHiNO3.. 10. 66 0.02 NHiNOL- 4. 11. 25 0.07 NHaNOa. .do 5. 33 13. 35 None Size 2/0 L... 3.82 10.23 Do Size 2/0 Chromie..- 4. 49 11. 76

Tanned with 2,4-dihydroxybenitole acid (1% solution), aluminum iormoacetate (1% solution), and formaldehyde (0,25% solution).

Example VI Collagen sutures are prepared by the indirect spinning process as described in U.S. Patent No. 3,114,372. The efiect of the addition of sodium chloride to the collagen dispersion as shown in Table 6.

I Size 5 0 ChIOmlG..- 1. 09

Example VII Collagen dispersions are prepared by swelling tendon collagen fibrils in dilute lactic acid (pH 3.0). To one half of this dispersion is added 0.5% potassium chloride. Sutures prepared from this collagen dispersion containing the potassium chloride by the indirect spinning process described in U. S. Patent No. 3,114,372 have a higher dry knot tensile strength than those prepared by the same procedure from the collagen dispersion containing no added potassium chloride.

A statistical analysis of a large number of controlled experiments provide a comparison between collagen dispersions containing an added electrolyte and those dispersions that do not contain an added electrolyte and demonstrate that extruded collagen strands have a higher dry knot tensile strength and dry straight tensile strength when a small amount of an electrolyte is present in the dispersion of collagen fibrils that is extruded.

While the invention has been described in detail according to the preferred manner of carrying out the process and yielding the products, it will 'be obvious to those skilled in the art, after understanding the invention, that changes and modifications may be made therein without de arting from the spirit or scope of the invention, and it is intended in the appended claims to cover such changes and modifications.

What is claimed is:

1. A dispersion of swollen undegraded collagen fibrils having a solids content of the order of 0.72% to 1.1% in a dispersant consisting essentially of an aqueous solution of an organic acid characterized by a pKa value greater than 2.40 and less than about 6.3; said aqueous acid solution having an acid content from about 0.2% to about 1.0% of the total weight of the solution, and containing from 0.01% to 0.0 7% of an added watersoluble salt.

2. The dispersion of claim 1 wherein the aqueous acid solution is a solution of cyanoacetic acid.

3. A dispersion of swollen undegraded collagen fibrils having a solids content of the order of 0.72% to 1.1% in a dispersant consisting essentially of an aqueous solution of an organic acid characterized by a pKa value greater than 2.40 and less than about 6.3; said aqueous solution, having an acid content from. about 0.2% to about 1.0% of the total weight of the solution, and con t aining from 0.01% to 0.07% of a water-soluble salt.

4. A dispersion of swollen undegraded collagen fibrils having a solids content of the order of 0.72% to 1.1% in a dispersant consisting essentially of an aqueous methanol solution of cyanoacetic acid having an acid content from about 0.2% to about 1.0% of the total weight of the solution, and a Water-soluble sal-t, said methanol amounting to. about 50% of the total weight of the dispersant and the salt being present in an amount that constitutes from 0.01% to 0.07% by weight of the dispersant.

A dispersion of swollen undegraded collagen fibrils having a solids content of the order of 0.72% to 1.1% in a dispersant consisting essentially of an aqueous methanol solution of cyanoacetic acid and a water-soluble salt, said methanol amounting to about 50% of the total weight of the dispersant, said salt amounting to between about 0.01% and 0.07% of the total weight of the dispersant, and said cyanoacetic acid amounting to between about 0.2% and about 0.5% of the total weight of the dispersant.

6. In a process for the extrusion of a homogeneous collagen dispersion having a solids content of the order of 0.72% to 1.1% to form a shaped article, the improvement which comprises swelling and dispersing collagen fibrils derived from animal tendons in an aqueous solution of an organic acid characterized by a pKa value greater than 2.40- and less than about 6.3; said acid solution having an acid content from about 0.2% to about 8 1.0% of the total weight of the solution, and having dissolved therein from 0.01% to 0.07% by Weight of water-soluble salt.

7. The process of claim 6 wherein the aqueous acid solution is a solution of cyanoacetic acid.

8. In a process for the extrusion of a homogeneous collagen dispersion having a solids content of the order of 0.72% to 1.1% to form a shaped article, the improvement which comprises swelling and dispersing collagen fibrils derived from animal tendons in an aqueous methanol solution of cyanoacetic acid having an acid content from about 0.2% to about 1.0% of the total weight of the solution, and having dissolved therein from 0.01% to 0.07% by weight of a water-soluble salt while maintaining the temperature below about C., said methanol amounting to about of the total weight of the aqueous methanol solution.

References Cited UNITED STATES PATENTS 2,039,262 4/1936 Schulte 106-124 2,845,362 7/1958 Jenkins et al 264202 3,114,593 12/1963 Griset et al. 156-296 ALEXANDER H. BRODMERKEL, Primary Examiner.

T. MORRIS, Assistant Examiner. 

